Overcoming the On‐Target Toxicity in Antibody‐Mediated Therapies via an Indirect Active Targeting Strategy

Abstract Antibody‐based therapies could be led astray when target receptors are expressed on nontarget sites, and the on‐target toxicity poses critical challenges to clinical applications. Here, a biomimetic indirect active targeting (INTACT) strategy is proposed based on receptor expression disparities between nontarget sites and the targets. By prebinding the antibodies using cell membrane vesicles with appropriate receptor expressions, the INTACT strategy could filter out the interactions on nontarget sites due to their inferior receptor expression, whereas ensure on‐demand release at the targets by competitive binding. The strategy is verified on CD47 antibody, realizing drastic alleviation of its clinically concerned hematotoxicity on a series of animal models including humanized patient‐derived xenograft platforms, accompanied by preferable therapeutic effects. Furthermore, the INTACT strategy proves extensive applicability for various systems including antibody, antibody–drug conjugate, and targeted delivery systems, providing a potential platform refining the specificity for frontier antibody‐related therapies.

# These authors contributed equally to this work. * Corresponding author. Email: chongli@swu.edu.cn; tp1232000@sina.com Figure S1. Quantitative analysis of CD47 expression on different cells. Figure S2. Quantification of total phospholipid content in CM. Figure S3. Quantitative counting of different nanoparticles and efficiency of fluorescein-labeling. Figure S4. Characterization of CM vesicles, NP, PCM@NP, and Anti-CD47-PCM@NP. Figure S5. Transmission electron micrographs of NP, PCM@NP, and Anti-CD47-PCM@NP. Figure S6. Determination of antibody loading on the surface of Anti-CD47-PCM@NP. Figure S7. The binding affinity of antibody with CM measured by surface plasmon resonance. Figure S8. Representative fluorescent images of uptake of NP, PCM@NP, and Anti-CD47-PCM@NP by RAW264.7 macrophages and 4T1 cells. Figure S9. Analysis of the expression level of CD47 in CD47 -/-4T1 cells. Figure S10. Appearance of the Real-time Single cell Multi-modal Analyzer. Figure S11. In vivo and ex vivo targeting ability of NP and PCM@NP in tumorbearing mice models. Figure S12. Representative fluorescent images in tumor, heart, lung, liver, spleen and kidney slices after different treatments. Figure S13. Pharmacokinetic analysis of Anti-CD47-PCM@NP. Figure S14. Representative images and phagocytic index of C57BL/6 bone marrowderived macrophages phagocytosing tumor cells after treatment. Figure S15. Ki67 percentage and immune cell infiltration in tumor sections after Anti-CD47-PCM@NP treatment, by immunohistochemistry and immunofluorescence staining. Figure S16. Expression of immune signatures for the identification of tumorinfiltrating immune cell. Figure S17. Construction of HuNSG mouse model bearing PDX tumor. Figure S18. Anti-tumor efficacy study on HuNSG PDX tumor-bearing mice. Figure S19. Body weight curves during the Anti-CD47-PCM@NP treatment. Figure S20. H&E staining of the main organs in mice treated with Saline, PCM@NP, Anti-CD47, Anti-CD47-PCM@NP. Figure S21. C. albicans infection status in mice kidney after different treatments. Figure S22. Characterization of NP/PTX, PCM@NP/PTX, and Anti-CD47-PCM@NP/PTX. Figure S23. The INTACT strategy enabled the co-delivery of antibody with smallmolecule chemotherapeutic agent for combinational therapy with precise targeting. Figure S24. Ki67 percentage and immune cell infiltration in tumor sections after Anti-CD47-PCM@NP/PTX treatment, by immunohistochemistry and immunofluorescence staining. Figure S25. Ki67 percentage and immune cell infiltration in tumor sections after ADC-PCM@NP treatment, by immunohistochemistry and immunofluorescence staining. Figure S26. Body weight curves during the ADC-PCM@NP treatment. Figure S27. H&E staining of the main organs in mice treated with Saline, ADC and ADC-PCM@NP. Figure S28. Characterization of CMV-Anti-CD47-Lip and functional analysis of the indirect active targeting in vitro. Figure S29. Flow cytometric analysis of the HER2 expression on the surface of TUBO and 4T1 cells. Figure S30. AST, CTN-I, LDH indexes, and H&E staining of hearts and lungs of mice after Anti-HER2-PCM@NP treatments. Table S1. Characterization of the CM vesicles, PLGA NPs and PLGA NPs loaded with PTX. Table S2. Antibody and metal isotopes for CyTOF. Table S3. The expression of specific markers of each cluster. Table S4. Hematotoxicity analysis of Anti-CD47. Table S5. In vitro anticancer activity test of Anti-CD47-PCM@NP/PTX and other formulations. Table S6. Hematotoxicity analysis of ADC. Movie S1. Instrumental field of view for single cell fluorescence signal detection by the Real-time Single cell Multi-modal Analyzer nanoprobe. Movie S2. Microscopic field of view for single cell fluorescence signal detection by the Real-time Single cell Multi-modal Analyzer nanoprobe. Movie S3. The binding of free Anti-CD47 to the target cells adsorbed in the cavity of the microfluidic chip in the flowing state.

Supplementary Materials list
Movie S4. In vitro transfer of CD47 antibody from Anti-CD47-PCM@NP to the surface of 4T1 cells was assessed by microfluidic assay. Movie S5. No significant surface binding of Anti-CD47 or PCM@NP internalization was observed on CD47 -/-4T1 cells.      Table S4. Hematotoxicity analysis of Anti-CD47 (Day 15).    The target cells (4T1) were adsorbed in the cavity of the microfluidic chip, and then exposed to FITC-labeled free Anti-CD47

RBC
in flowing state, and the binding of Anti-CD47 on 4T1 cells was recorded in real-time by a fluorescence microscope. The video shows that Anti-CD47 could bind to the target cells.
Movie S4. In vitro transfer of antibody from Anti-CD47-PCM@NP to the surface of 4T1 cells was assessed by microfluidic assay.
The target cells (4T1) were adsorbed in the cavity of the microfluidic chip, and then exposed to Anti-CD47-PCM@NP in a flowing state (antibody was labeled with FITC, and PCM@NP was labeled with DiD), and the fluorescence was recorded in realtime by the fluorescence microscope. The video shows that antibodies were effectively released from Anti-CD47-PCM@NP and accumulated around the target cells membrane, accompanied by substantial internalization of the dissociated PCM@NP.

Movie S5. No significant surface binding of antibody or PCM@NP internalization was observed on CD47 -/-4T1 cells.
The non-target cells (CD47 -/-4T1) were adsorbed in the cavity of the microfluidic chip, and exposed to the Anti-CD47-PCM@NP in a flowing state (antibody was labeled with FITC, and PCM@NP was labeled with DiD), and the fluorescence microscope was recorded in real-time. The video shows that no significant surface binding of antibody or PCM@NP internalization was observed on non-target cells.